Project Summary Pancreatic ductal adenocarcinoma (PDAC) is an aggressive and poorly understood disease with the worst 5- year survival of all cancers (5-year survival of 7%). Although most patients are diagnosed at Stage IV, even patients who are diagnosed at Stage I have the poorest survival of all Stage I cancers (5-year survival 26%). PDAC is one of the few cancers with no targeted therapies, and currently available chemotherapies only improve survival by 6 months. This poor outcome is due in part to the dense stroma that inhibits drug delivery, metabolically cooperates with the tumor to sustain growth, promotes immunosuppression, and sends mechanical cues to the tumor to promote invasion. Therefore, new drug development for PDAC focuses on combination treatments that alter the stromal microenvironment for improved drug efficacy. This remains an unsolved problem as mouse models to test candidate drugs in vivo suffer from high cost, low throughput, slow read out time, and limited relevance to the human condition. Conversely, high-throughput screens of drugs are performed in tumor cells alone in monolayer, which does not account for the critical role of the stroma in PDAC drug response. The goal of this proposal is to develop a relevant 3D in vitro model of human PDAC that incorporates both the cells in conjunction with the corresponding biomimetic stroma. We will create the in vitro model system through multiphoton excited fabrication, which affords the synthesis of 3D printed scaffolds with submicron resolution and with extracellular matrix (ECM) proteins, e.g. collagen. We will derive the designs directly from high resolution Second Harmonic Generation (SHG) images of the collagen in normal and malignant pancreatic stroma. This novel system will enable drug screens of human PDAC cells and stromal cells (including primary patient-derived cells) in a relevant human collagen matrix, thus increasing the information content of high-throughput pre-clinical drug screens. This initial project will build and validate the in vitro model of PDAC; measure cell response (migration, proliferation, metabolism) in co-culture (human epithelial and fibroblasts lines); and measure the efficacy to standard-of-care chemotherapies, and novel targeted therapies. We propose the following Aims: Aim 1. Build and validate 3D extracellular matrix models based on SHG images of collagen architecture of normal pancreas and PDAC using multiphoton excited fabrication. Aim 2. Measure changes in cell migration, proliferation, and metabolism in response to normal and tumor matrix. Aim 3. Measure treatment response in normal and tumor matrix conditions.